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Neurochemical Research, Vol. 28, No. 12, December 2003 (© 2003), pp. 1793–1797

Ketogenic Diet Increases Glutathione Peroxidase Activity


in Rat Hippocampus

Denize R. Ziegler,1 Leticia C. Ribeiro,2 Martine Hagenn,3 lonara R. Siqueira, 2


Emeli Araújo,2 Iracy L. S. Torres, 2 Carmem Gottfried, 1,2 Carlos Alexandre Netto,2
and Carlos-Alberto Gonçalves2

(Accepted April 23, 2003)

Ketogenic diets have been used in the treatment of refractory childhood epilepsy for almost
80 years; however, we know little about the underlying biochemical basis of their action. In
this study, we evaluate oxidative stress in different brain regions from Wistar rats fed a ketogenic
diet. Cerebral cortex appears to have not been affected by this diet, and cerebellum presented
a decrease in antioxidant capacity measured by a luminol oxidation assay without changes in
antioxidant enzyme activities—glutathione peroxidase, catalase, and superoxide dismutase. In
the hippocampus, however, we observed an increase in antioxidant activity accompanied by an
increase of glutathione peroxidase (about 4 times) and no changes in lipoperoxidation levels.
We suggest that the higher activity of this enzyme induced by ketogenic diet in hippocam-
pus might contribute to protect this structure from neurodegenerative sequelae of convulsive
disorders.

KEY WORDS: Ketogenic diet; oxidative stress; glutathione peroxidase; lipoperoxidation; hippocampus.

INTRODUCTION formation as a normal by-product of their activity. Sev-


eral regions of the brain are particularly rich in iron,
Brain tissue is particularly vulnerable to oxidative which promotes the production of damaging oxygen
damage, possibly because of its high consumption of free radical species. Furthermore, the brain is relatively
oxygen and the consequent generation of high quantities poorly endowed with protective antioxidant enzymes or
of reactive oxygen species (ROS) during oxidative phos- antioxidant compounds (2,3).
phorylation (1). Moreover, several enzymes expressed in ROS formation has been implicated in damage to
brain, including monoamine oxidase, tyrosine hydroxy- cerebral tissue in several nervous pathologies, such as
lase, and L-amino acid oxidase, lead to hydrogen peroxide ischemia-reperfusion injury, Parkinson’s disease, and
epilepsy (4). Active oxygen species concentrations are
1
often increased during seizure activity, and both initia-
Centro de Ciências da Saúde, Universidade do Vale do Rio dos Sinos,
São Leopoldo, RS, Brazil.
tion and propagation of lipid peroxidation have been sug-
2
Departamento de Bioquìmica, ICBS, Universidade Federal do Rio gested to play a role in epileptogenesis (5–7).
Grande do Sul, Porto Alegre, RS, Brazil. The ketogenic diet (KD) has been used in the treat-
3
Departamento de Fisiologia, ICBS, Universidade Federal do Rio Grande ment of refractory childhood epilepsy since the early
do Sul, Porto Alegre, RS, Brazil. 1920s and reemerged as an important alternative clini-
4
Address reprint requests to: Denize Righetto Ziegler, Centro de
Ciências da Saùde, Unisinos, Av. Unisinos, 950–Caixa postal: 275,
cal approach in the 1990s. The efficacy of the diet as a
93022-000, São Leopoldo RS, Brazil. Fax: 55-51-590 8122; E-mail: treatment for human epilepsy has been suggested by
denize@bios.unisinos.br clinical evidence (8,9). The KD is designed to stimulate
1793
0364-3190/03/1200–1793/0 © 2003 Plenum Publishing Corporation
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1794 Ziegler et al.

the biochemical effects of fasting by maintaining a state Table I. Composition of the Control and Ketogenic Diets
of ketosis, the resulting condition provides for much of
Control diet* g/100 g Ketogenic diet g/100 g
the cerebral energy requirements in the form of ketone
bodies. Despite the clinical success, there have been Total fat 11 Lard 69
remarkably few studies pointing to the possible mechan- Sunflower oil 0.5
Protein 22 Protein† 24
isms of that diet, as well as to their effects on distinct Fiber 3 Fiber 1
aspects regarding central nervous system metabolism Ash 6 Ash‡ 4
(9,10). Vitamin 2 Vitamin¶ 1.5
Carbohydrates 52 Carbohydrates 0
It should be interesting to investigate whether KD
changes brain antioxidant activities. In this study we *Commercial nonpurified diet, Nuvilab-CR1 (Curitiba, Brazil).
evaluate oxidative stress in different brain regions (hip- †
Casein, purity 87% (from Herzog, Porto Alegre, Brazil) supplemented
pocampus, cerebral cortex, and cerebellum) from rats fed with 0.15% L-Methionine (from Merck, Rio de Janeiro, Brazil).

Mineral mixture (from Roche, São Paulo, Brazil), mg/100 g of ration:
a KD. We measured lipid peroxidation assayed by levels NaCl, 557; Kl, 3.2; KH2PO4, 1556; MgSO4, 229; CaCO3, 1526;
of thiobarbituric acid reactive substances (TBARS), total FeSO4.7H2O, 108; MnSO4.H2O, 16; ZnSO4.7H2O, 2.2; CuSO4.5H2O,
antioxidant reactivity (TAR) estimated through measure- 1.9; CoCl2.6H2O, 0.09.

Vitamin mixture (from Roche, São Paulo, Brazil), mg/100 g of ration:
ments of luminol oxidation assay, and antioxidant enzyme vitamin A, 4; vitamin D, 0.5; vitamin E, 10; menadione, 0.5; choline,
activities—catalase, superoxide dismutase and glutathione 200; PABA 10; inositol 10 mg; niacin, 4; pantothenic acid, 4; riboflavin,
peroxidase. 0.8; thiamine, 0.5; pyridoxine, 0.5; folic acid, 0.2; biotin, 0.04; vitamin
B12, 0.003.

EXPERIMENTAL PROCEDURE
luminol luminescence, calculated as the ratio lo/l, where lo is the
Reagents and Equipment. All chemicals were purchased from luminescence intensities in the absence of additives and l
Sigma (St. Louis, MO, USA) except for the RANSOD kit, which was is the luminescence intensity after addition of a small aliquot of
purchased from RANDOX. Total antioxidant capacity was assayed using the sample. A comparison of the ratio lo/l of Trolox (20 nM) and
a beta liquid scintillation spectrometer (Wallac model 1409), and the the samples allows obtaining TAR values as equivalents of Trolox
enzyme activities were measured with a double-beam spectrophotome- concentration.
ter with temperature control (Hitachi U-2001). Catalase (CAT) Assay. CAT activity was assayed by the method
Animals and Diet. Male 30-day-old Wistar rats came from the of Aebi (14), which is based on the disappearance of H2O2 at 240 nm.
local breeding colony (ICBS-UFRGS). Animals were weight matched Brain tissue was homogenized 1:10 (w/v) in 10 mM potassium phos-
and divided into two groups: control rats that received regular labo- phate buffer, pH 7.6. One unit is defined as one M of hydrogen
ratory chow (Nuvilab-CR1, Nuvital, Brazil) and treated rats that peroxide consumed per minute, and the specific activity is reported
received a KD (Table I) for 8 weeks (10). They were maintained in as units per milligram of protein.
a ventilated room at 21°C, with free access to food and water on a Superoxide Dismutase (SOD) Assay. The assay for SOD activ-
12-h light/dark cycle. All animal procedures were in accordance with ity was carried out with the RANSOD kit (Randox, USA). Cerebral
the NIH guidelines for the care and use of laboratory animals and tissue was homogenized 1:10 (w/v) in 10 mM potassium phosphate
were approved by the local authorities. buffer, pH 7.4. This method is based on the formation of red
Tissue Preparation. Animals were killed by decapitation, and the formazan from the reaction of 2-(4-iodophenyl)-3-(4-nitrophenol)-
brain regions were dissected on ice. Brain tissue was homogenized in 5-phenyltetrazolium chloride (INT) and superoxide radical (pro-
the incubation medium used for each technique and centrifuged at duced in the incubation medium from xanthine oxidase reaction),
1000  g for 10 min at 4°C, and the supernatant was immediately which is assayed in a spectrophotometer at 505 nm. The inhibition
used for the lipid peroxidation and antioxidant reactivity measure- of the produced chromogen is proportional to the activity of the
ments. For the enzyme activity determinations, the brain tissue was SOD present in the sample. A 50% inhibition is defined as 1 unit
kept frozen at 70°C for up to 1 week. of SOD, and specific activity is expressed as units per milligram of
Thiobarbituric Acid Reactive Substances (TBARS) Assay. TBARS protein.
were determined immediately after tissue homogenization by a fluores- Glutathione Peroxidase (GPx) Assay. GPx activity was measured
cence method (11). After extraction with n-buthanol, fluorescence was by the method of Wendel (15), except for the concentration of NADPH,
measured at 515 nm excitation and 555 nm emission. Values were which was adjusted to 0.1 mM. Tissue was homogenized 1:10 (w/v) in
expressed as nM TBARS/g tissue, using malondialdehyde standards pre- 10 mM potassium phosphate buffer, pH 7.6. Tert-butyl-hydroperoxide
pared from 1,1,3,3-tetramethoxypropane. was used as substrate. NADPH disappearance was monitored with a
Total Antioxidant Reactivity (TAR) Assay. The method was spectrophotometer at 340 nm. One GPx unit is defined as 1 Mol of
based on Lissi et al. (12) and Desmarchelier et al. (13). The reac- NADPH consumed per minute, and specific activity is reported as units
tion mixture contains the free radical source 2 mM 2,2 azobis per milligram of protein.
(2-amidopropane (ABAP) and 6 mM luminol in glycine buffer Protein Determination. Protein was measured by the method of
(0.1 M, pH 8.6). Incubation of this mixture at 20°C generates an Lowry et al. (16) using bovine serum albumin as standard.
almost constant light intensity that was measured in a scintillation Statistical analysis. Results are expressed as mean values  SEM.
counter (Beckman) working in the out of coincidence mode. The Student’s t test was used to analyze the significance of differences
TAR values were determined by measuring the initial decrease of between control and experimental groups.
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Ketogenic Diet Increases Glutathione Peroxidase Activity 1795

RESULTS

Thirty-day-old rats fed a KD for 8 weeks gained


weight similarly to controls. We used a semiquantitative
method for evaluate ketonemia at sacrifice day. Ketonemia
in control rats was less than 0.4 mM and in ketogenic rats
was about 2.4 mM.
Brain regions examined showed distinct behavior
in regard to TBARS levels (Fig. 1). In cerebral cortex
and hippocampus, TBARS levels were similar between
KD-treated animals and controls, suggesting that lipo-
peroxidation within those structures was not altered.
However, TBARS levels were significantly increased
in cerebellum of KD-treated rats, indicating an increase
in lipoperoxidation resulting from the change in the diet Fig. 2. Effects of KD on TAR assay in rat hippocampus (Hc),
cerebral cortex (Cx) and cerebellum (Cb). TAR was measured by
composition. decrease of luminol luminescence. Results are expressed as
The analysis of total antioxidant capacity (TAR) percentage of control. Columns represent mean  SEM of eight
in cerebral cortex revealed no significant differences independent experiments performed in duplicate. The mean TAR
values (expressed as pM eq. Trolox/mg protein) from control group
between the two groups. However, rats treated with a KD were 57.37  9.8 (Hc), 70.42  1.3 (Cx), and 74.97  1.2 (Cb).
had a significant increase in their antioxidant capacity in *Values significantly different from control group, as determined by
hippocampus and a significant decrease in the cerebellum Student’s t test (P  0.05).
(Fig. 2).
We found changes of TBARS and TAR in hippo-
campus and cerebellum, and therefore we decided to inves- active, around 400%, in KD-fed animals, although SOD
tigate antioxidant enzyme activities in these regions. There activity did not vary between groups (Table II).
were no significant differences between experimental
groups regarding the three enzymes’ activities—superoxide
dismutase (SOD), catalase (CAT), and glutathione peroxi- DISCUSSION
dase (GPx) in cerebellum. However, in hippocampus, CAT
activity decreased around 50% and GPx was much more The understanding of possible mechanisms that
underlie the therapeutic effects of KD in epileptic dis-
orders is very important. It has been widely suggested
that nutritive dietary constituents can promote or hinder
the development of several chronic diseases (17), mainly
because of an increased susceptibility to or protection
against free radicals, respectively. This is, to our know-
ledge, the first study to focus on the relationship between
KD and oxidative stress in CNS.
Oxidative stress has been defined as the increase in
steady-state concentrations of active oxygen species,
either resulting from an overproduction of radical species
and/or as a consequence of antioxidant defenses deple-
tion. It has been widely recognized that the susceptibility
to oxidative stress differs according to specific brain
region (18–20). According to this characteristic, oxida-
tive stress has distinct effects in the brain structures
Fig. 1. Effects of KD on TBARS levels in rat hippocampus (Hc), studied. Cerebral cortex seems to have not been
cerebral cortex (Cx), and cerebellum (Cb). Wistar rats were fed with
control or ketogenic diet (KD). TBARS levels were measured at affected by KD, maintaining the lipoperoxidation level
515-nm excitation and 555-nm emission wavelengths. Columns or the total antioxidant capacity. However, in KD rat
represent mean  SEM of six independent experiments performed in cerebellum, there was a decrease in antioxidant capa-
duplicate. Student’s t test was used to evaluate the significance of
differences between paired group means. city not resulting from a drop in antioxidant enzyme
*Values significantly different from control diet group (P  0.01). activities. Increased lipoperoxidation may be due to a
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1796 Ziegler et al.

Table II. Effects of Treatment with KD on Enzyme Activities in Hippocampus and Cerebellum
Homogenates from Rats

Enzymes activities (units/mg protein)

Groups CAT SOD GPx

Hippocampus control 0.120  0.016 56.84  3.14 3.39  0.41


Hippocampus KD 0.068  0.005* 51.31  2.56 13.60  1.40*
Cerebellum control 1.720  0.190 71.40  6.42 9.52  0.85
Cerebellum KD 1.720  0.240 66.60  3.03 11.02  1.65

Note: Results are mean  SEM of eight independent experiments performed in duplicate. One CAT unit is
defined as 1 M of H2O2 consumed per minute. One SOD unit is defined as 50% inhibition of red for-
mazan formation. One GPx unit is defined as 1 M of NADPH consumed per minute.
*Values significantly different from diet control group as determined by Student’s t test (P  0.05).

higher susceptibility of this structure to the effects of oxidative stress (27). However, at this moment there is no
ROS caused by the change in diet composition. The evidence relating degree of fatty acid unsaturation in the
hippocampus, however, showed an opposite profile. We several KD formulas and efficacy of this diet in epileptic
have observed an increase in antioxidant defense capa- disorders.
city, and, probably associated to that fact, there was no
change in lipoperoxidation. This increase was due, at
least partially, to an increase in the activity of the antioxi-
CONCLUSION
dant GPx enzyme, that might have been stimulated by
several factors, among them an increase in ROS pro-
There are many hypotheses about how a KD can
duction itself. The increase in GPx activity was so impor-
affect epileptic diseases. The relationship between
tant as to guarantee protection, even though CAT activity
free radical and scavenger enzymes with epilepsy has
decreased. GPx appears to play a major role in metaboliz-
been found, and ROS have been implicated in seizure-
ing hydrogen peroxide in neural tissue (21,22). We do
induced neurodegeneration (see Schwartzkroin [28] for
not know whether these effects are caused by high circu-
a review).
lating levels of ketone bodies or by the lipid components
Our data suggest that ketogenic diet maybe protec-
of a KD.
tive in epileptic disorders by affecting antioxidant activ-
Ketone bodies are able to affect oxidative stress in
ity, particularly that of GPx. Supporting that, a reduced
nonneural cells. Cultures of polymorphonuclear leukocytes
intracellular GPx activity in children resistant to con-
and red blood cells from healthy subjects exposed to ketone
ventional pharmacological therapy, the main indication
bodies presented a reduced production of superoxide (23)
of KD, has been reported (29). Additionally, in the rat
and accumulation of oxidized glutathione, respectively model of epilepsy induced by pilocarpine, an increase of
(24). Acetoacetate, but not beta-hydroxybutyrate, increased
hippocampal GPx during the first hour of status epilep-
lipid peroxidation in cultured human umbilical vein
tic was observed (30). A high activity of GPx induced
endothelial cells (25). Further studies in cultured neural
by KD in hippocampus might contribute to protect this
cells from different brain regions will be useful to detail
structure from the neurodegenerative sequelae of epilep-
our results and to characterize the possible direct effect of
tic disorders.
ketone bodies.
Another possibility to explain the differences that we
found would be conceiving that the lipid component of a
KD could change lipid composition of the membranes ACKNOWLEDGEMENTS
and/or cellular antioxidant activity. For example, changes
in fatty acid unsaturation of mitochondria membranes are Supported by Brazilian funds from Conselho Nacional de Desen-
accompanied by changes in the susceptibility and gener- volvimento Científico e Tecnológico (CNPq), PRONEX (66.136/
1996-0) and Fundação de Amparo a Pesquisa do Rio Grande do
ation of reactive oxygen species (26). Moreover, polyun- Sul (FAPERGS). The authors are very grateful to Dr. Adriana Bello
saturated fatty acids could play a role by direct control of Klein and Dr. Suzana Lores Arnaiz for their comments on the
gene expression in many neurological diseases involving manuscript.
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Ketogenic Diet Increases Glutathione Peroxidase Activity 1797

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